Significance ? Breast cancer (BC) is one of the most common causes of cancer deaths for women with increasing incidence among women Veterans in the VA Health Care System. Patient survival has improved dramatically for primary BC but metastatic BC, for which targeted agents are usually not available, is common in African-American Veterans and is largely incurable. Most BC treatments target proliferating tumor cells that rely on glycolysis to fuel their metabolic needs. However, metastatic BC may exhibit a cancer stem cell phenotype with considerable dormancy and acquired drug resistance. These BC, including triple negative BC (TNBC), are often dependent on mitochondrial respiration (oxidative phosphorylation; oxphos) to generate energy and promote survival. Since there are no targeted therapies for TNBC and since most mitochondrial-targeting drugs exhibit substantial toxicity, there is a need to find new and safer therapeutic agents. Using a direct drug discovery approach and computer-assisted drug design (CADD), we identified novel small molecules that interfere with protein:DNA binding and transcriptional activity. While normal epithelial cells were relatively resistant, a lead compound (CADD522) inhibited BC cell proliferation and tumorsphere formation, delayed tumor growth and metastasis in vivo, and inhibited mitochondrial adenosine triphosphate (ATP) synthase and respiration (oxygen consumption) while increasing the levels of reactive oxygen species (ROS). Premise ? Understanding the molecular mechanisms of targeting mitochondrial ATP synthase to elevate ROS in cancer cells will likely result in novel therapeutics against metastatic BC. Patients with drug-resistant, dormant or metastatic disease could benefit from a therapeutic approach that targets mitochondrial oxphos by inhibiting ATP synthase. Therefore, we propose the hypothesis that targeting mitochondrial metabolism with a novel ATP synthase inhibitor will inhibit BC tumor progression and metastasis by lowering ATP levels, reducing cellular respiration, and increasing ROS damage for therapeutic benefit. Specific Aims ? Specific Aim 1: Define the mechanistic basis for CADD522-mediated ATP synthase inhibition in restraining BC tumor cell proliferation. Mitochondrial oxygen consumption rate (OCR), global gene expression profiles and direct targeting of ATP synthase will be defined. Specific Aim 2: Determine the mechanisms through which CADD522-mediated OCR inhibition increases reactive oxygen species (ROS) to reduce glucose utilization. Redox balance, pyruvate dehydrogenase (PDH) activity, and TCA cycle flux will be measured. Specific Aim 3: Define the translational potential of mitochondrial targeting with CADD522 to promote ROS damage and inhibit BC growth and metastasis. In vitro toxicological assays and in vivo tumor models will assess translational potential after oral administration of a novel therapeutic agent. Overall Impact ? Elucidating how reprogrammed cancer cell metabolism promotes BC progression may lead to strategies to prevent or treat metastatic BC. Using agents that target a tumor?s metabolic requirements is an innovative approach and may inhibit metastatic pathways involving mitochondrial metabolism (stem-like and/or slow-growing tumors). These mitochondria-targeted approaches will exploit differences between normal and cancer cells, which may ultimately have an impact on clinical efficacy and safety. Discovery of new metabolic biomarkers will aid in patient stratification and clinical evaluation thus providing strong justification for future investigational new drug development. In summary, these approaches will be fundamental in elucidating the translational potential of metabolic targeting, are of relevance to the VA health care mission, and will likely lead to the discovery of new treatments for Veterans with metastatic BC.